1. Field of the Invention
This invention relates to enhanced luminescence of covalently coupled dyes containing lanthanide macrocycle complexes, especially as tags for detecting members of combining pairs, and to the detection of low levels of these dyes.
2. Prior Art
The sensitivity of fluorescence measurements for the analysis of biological samples is often limited by background signal due to autofluorescence or Raman scattering.
At present, the microscopic visualization of luminescent labels containing lanthanide (III) ions, primarily europium (III), as light emitting centers is best performed with time-gated instrumentation, which by virtually eliminating the background fluorescence, results in an improved signal to noise ratio. Although the use of time-gated luminescence for microscopic and clinical chemistry analyses holds the promise of maximizing the detectability and quantitation of markers containing lanthanide complexes, this instrumentation is costly and not widely available; furthermore, time-gated measurements often involve loss of signal or precision. The combination of an image-intensifier coupled to a CCD permits the high speed gating of image acquisition. Briefly, rapid voltage changes at the intensifier dynodes result in concomitant changes in the current amplification, which effectively shutters the photoelectrons impinging on the phosphor that is located directly in front of the CCD surface. Other time-gating approaches to control the image acquisition of digital microscopes include high speed rotating choppers and ferro-electric shutters. However, none of these is suitable for the clinical pathology or clinical chemistry laboratory.
Vallarino and Leif U.S. Pat. Nos. 5,373,093 and 5,696,240 disclosed hexa-aza-macrocyclic complexes incorporating a lanthanide, actinide, or yttrium ion possessing high kinetic stability and pendant functional groups that can be readily coupled/conjugated to a biologically active molecule such as an antibody or antigen, or to a biologically compatible ionically uncharged macromolecule such as a linear or cross-linked polysaccharide. The complexes of europium (III) and terbium (III), for example, possess a long-lived fluorescence intensity that can be substantially increased by inter-action with a suitable enhancer, for example the sodium salt of dibenzoylmethane or thenoyltrifluoroacetyl-acetone. The entire disclosure of these patents is here incorporated by reference.
Xu U.S. Pat. No. 5,316,909 disclosed the interaction between B-diketonate complexes of luminescent lanthanide (III) and yttrium (III) in the presence of a synergistic compound to provide a cofluorescence effect which significantly increases the emission intensity. The embodiment described requires that the original chelate containing the fluorescent ion be dissociated from the biomolecule, followed by the addition of the yttrium cofluorescence species and incubation with the synergistic compounds for 1 to 15 minutes prior to measurement.
Adeyiga et al. SPIE vol. 2678, pages 212-220 (1996) disclosed extension of Xu""s method to other lanthanide complexes including the europium (III) macrocycles. It is stated at page 215 that xe2x80x9ca preliminary, unoptimized study with the parent unfunctionalized macrocycle {Eu(C22H26N6)} triacetate complex as prototype has shown an approximate three fold increase in luminescence.xe2x80x9d
In accordance with this invention, there is provided a spectrofluorimetrically detectable luminescent composition comprising water, a micelle-producing amount of at least one surfactant, at least 1xc3x9710xe2x88x9210 moles/liter of at least one energy transfer acceptor lanthanide element macrocycle compound having an emission spectrum peak in the range from 500 to 950 nanometers, and a luminescence-enhancing amount of at least one energy transfer donor compound of yttrium or a 3-valent lanthanide element having atomic number 59-71, provided that the lanthanide element of said macrocycle compound and the lanthanide element of said energy transfer donor compound are not identical.
The enhanced luminescence of compositions according to the invention permits the detection and/or quantitation of the lanthanide macrocycle compound and complexes thereof without the use of expensive and complicated time-gated detection systems. As a result, these macrocycle compounds and complexes thereof are useful as reporter molecules in immunoassays, analytical cytology, histological staining, and imaging processing.
The enhanced luminescence of compositions according to the invention caused by a different lanthanide energy transfer donor compound can also occur with functionalized derivatives of energy transfer acceptor lanthanide macrocycles, that is macrocycles substituted with reactive functional groups at which reaction with analytes can take place; with reaction products of such functionalized macrocycles with such analytes; and with polymers which contain multiple lanthanide-containing units. Through their reactive functional groups, functionalized energy transfer acceptor lanthanide macrocycles can be attached by a coupling functionality to analytes including small molecules of biological interest having molecular weights from 125 to 2000 daltons, such as nucleic acid bases or haptens, and large molecules of biological interest having molecular weights greater than 2000 daltons such as proteins including antibodies, polysaccharides, or nucleic acids.
Also in accordance with this invention, there is provided a method for analysis of a sample suspected of containing at least one analyte, frequently a biologically active compound, said method comprising:
a) contacting said sample with a functionalized complex of a metal M, where M is a metal ion selected from the group consisting of a lanthanide having atomic number 57-71, an actinide having atomic number 89-103 and yttrium(III) having atomic number 39;
in a reaction medium under binding conditions, whereby said analyte when present either interacts with said complex to form a conjugate or competes for interaction with a binding material specific for interaction with said complex and with said analyte;
b) adding to said reaction medium a luminescence-enhancing amount of at least one energy transfer donor compound of yttrium or a 3-valent lanthanide element having atomic number 59-71, provided that the lanthanide element of said macrocycle compound and a lanthanide element of said energy transfer donor compound are not identical,
(c) subjecting said reaction medium to excitation energy in the range of 200-400 nm, whereby enhanced luminescence in the range of 500-950 nm is generated,
(d) monitoring said luminescence of the reaction medium to measure in said sample at least one of the following:
(1) presence and/or concentration of said conjugate;
(2) presence and/or concentration of the product of the interaction of said complex with said binding material; and
(3) presence and/or concentration of the product of the interaction of the conjugate with the binding material.
It is a feature of this invention that the method does not require prior dissociation of the luminescence-enhanced complex before measuring its emission spectrum. Moreover, since the excitation spectra of lanthanide macrocycles and those of several DNA-specific dyes, including 4xe2x80x2,6-diamidino-2-phenylindole (DAPI) occur in the same region of the ultraviolet, both types of compounds can be excited at the same wavelength, while their emission spectra are very different. The organic dyes have broad emissions in the blue region of the spectrum while the enhanced luminescence of lanthanide macrocycles according to this invention occurs as very narrow emission peaks in the red. This difference allows the major emission of the enhanced luminescence composition of this invention to be unambiguously detected even when its intensity is much lower than that of the very strong emission of the DNA specific organic dyes.
It is a further feature of the invention that the composition and method of the invention not only provide enhanced luminescence but also minimize the interfering effect of non-specific binding of lanthanide metal macrocyclic compounds and complexes to substrates.
The lanthanide energy transfer acceptor macrocyclic compound ingredient of the composition of the invention is characterized by kinetic stability even in very dilute aqueous solution. The compound is resistant to removal or exchange of the central metal atom, and has a counterion or balancing anion readily exchanged for other anions. The term xe2x80x9clanthanidexe2x80x9d is used throughout the specification and claims to refer to central atoms of yttrium (III) and 3-valent actinide atoms (atomic number 89-103) as well as to 3-valent central atoms of lanthanide elements of atomic number 57-71.
The lanthanide energy transfer acceptor macrocyclic compound ingredient of the composition of the invention is further characterized by the fluorescence spectrum with emission in the range from 500 to 950 nanometers upon excitation in the range from 200 to 400 nanometers.
The macrocycle of the lanthanide energy transfer acceptor macrocyclic compound has six coordinating atoms, of which at least 4 are nitrogen atoms, and the remainder are nitrogen, oxygen, or sulfur.
In particularly preferred compositions of the invention, the lanthanide energy transfer acceptor macrocyclic compound has the formula 
Wherein
M is a metal ion selected from the group consisting of a lanthanide having atomic number 57-71, an actinide having atomic number 89-103 and yttrium(III) having atomic number 39;
R is a substituent selected from the group consisting of hydrogen, straight-chain alkyl, or branched-chain alkyl; aryl-substituted alkyl, aryl, and alkyl-substituted aryl, with the proviso that such substituent does not limit the solubility of the resultant complex or otherwise interfere with the cyclization of such complex during its synthesis;
X is selected from the group consisting of nitrogen, sulfur and oxygen which forms a part of a ring structure selected from the group consisting of pyridine, thiophene or furan, respectively, at the positions marked X;
n is 2 or 3;
Y is a negatively charged ion, including acetate, carboxylate, sulfonate, halide, nitrate, perchlorate, thiocyanate, and picrate, with the proviso that such negative ion does not limit the solubility of the resultant complex or otherwise interfere with either the coupling procedure or the energy transfer leading to fluorescence;
m is the ionic charge of the metal ion in the macrocyclic complex, and;
y is the ionic charge of the counterion in the macrocyclic complex.
A, B, C, and D are selected substituents selected from the group consisting of hydrogen, straight-chain alkyl, or branched-chain alkyl; aryl-substituted alkyl, aryl, or alkyl-substituted aryl; reactive functionality, functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, or functionalized alkyl-substituted aryl;
Straight chain and branched chain alkyl substituents at A, B, C, and/or D have from 1 to 25 carbon atoms. Reactive functionality signifies any substituent capable of reacting with a compound of biological interest to form a covalent bond, such as alcoholic hydroxyl, phenolic hydroxyl, aldehyde, carboxylic acid, carboxamide, halogen, isocyanate, isothiocyanate, mercapto and nitrile substituents. Functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, and functionalized alkyl-substituted aryl signify the respective alkyl; aryl-substituted alkyl, aryl, and alkyl-substituted aryl groups substituted with a reactive functionality thereby spaced from the macrocycle as desired. Thus, a 10 carbon alkyl chain at A, B, C, and/or D can bear a terminal aminophenyl group, further illustrative functionalized substituents include hydroxymethyl, 4-hydroxybenzyl, 4-aminobenzyl, and 4-isothiocyanatobenzyl.
For convenience, the following abbreviations can be used to refer to compounds of Formula I. Any and all of the lanthanide ions including those having atomic number 57-71, actinides having atomic number 89-103 and yttrium (III) having atomic number of 39 are represented by M. Specific metal ions are represented by their standard chemical abbreviation. The generic term MMac refers to any and all of the macrocyclic species of Formula I. The unfunctionalized, mono-functionalized, and di-functionalized macrocyclic complexes of Formula I are abbreviated respectively as: xe2x80x9cMacxe2x80x9d, xe2x80x9cMac-monoxe2x80x9d and xe2x80x9cMac-dixe2x80x9d. When a specific peripheral pendant substituent having at least one reactive site (reactive functionality) is specified, its abbreviation is given as a suffix. Thus the compound of Formula I shown below, in which M is europium, each R is methyl (as shown by bond lines without termination) and each of A and B is a 4-isothiocyanatobenzyl group, is abbreviated as EuMac-di-NCS. The compound of Formula I shown below, in which M is terbium, each R is methyl, and B is a 4-isothiocyanatobenzyl group, is abbreviated as TbMac-mono-NCS, and the unfunctionalized compound of Formula I shown below, in which M is europium, each R is methyl and each of A and B is hydrogen, is abbreviated as EuMac. 
Formula II. Schematic formula of a di-functionalized europium macrocyclic complex. This structure is one of the isomers of the cationic europium macrocyclic moiety containing a 4-isothiocyanate-benzyl- substituent on each of the aliphatic side-chains. The molecular formula of the moiety is C38H36N5S2Eu. This and those that follow that include methyl groups adhere to the present convention of showing methyl groups as bond lines without termination. 
Formula III. Schematic formula of a cationic mono-functionalized terbium macrocyclic complex containing a 4-isothiocyanate-benzyl-substituent on one of the aliphatic side-chains, where the metal ion is terbium(III). The molecular formula of the moiety is C30H31N5STb. 
Formula IV. Schematic formula of a cationic unfunctionalized europium macrocyclic complex. This structure is the unfunctionalized prototype of the EuMac.
For the synthesis of these lanthanide element macrocycle compounds including access to the required starting materials, reaction conditions, purification, and subsequent coupling reactions with compounds of biological interest, reference can be had to Vallarino et al. U.S. Pat. Nos. 5,373,093 and 5,696,240 here incorporated by reference.
In a preferred group of compositions of this invention, at least one of the substituents A, B, C, and D of Formula I is a reactive functionality or a functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, or functionalized alkyl-substituted aryl group. Through these substituent groups, coupling or noncovalent binding can take place with an analyte, which can be a biologically active compound or any other compound able to interact with a functionalized substituent at A, B, C, and/or D.
Such coupling can take place directly, as in a conjugate of a MMac with a protein or a polynucleotide linked to the MMac through a functionalized group at A, B, C, or D.
Coupling of a functionalized group at A, B, C, or D with an analyte can also take place indirectly, by reaction of the functionalized group and a bridging/linking moiety providing the capability for derivatization with a receptor molecule or an entity for which there is a corresponding receptor molecule together with controlled spacing of the substrate of biological interest relative to the macrocycle of Formula I. Thus coupling is accomplished indirectly, either by the use of a bifunctional crosslinking reagent that provides covalent binding to the substrate of biological interest, or by binding the macrocycle to another molecule that has a high affinity for the substrate. To illustrate, avidin can couple with a functionalized macrocycle as well as with biotin, thus providing a link between biotin and the MMac. In another illustrative reaction, an amine-functionalized macrocyclic complex of Formula I is acylated with a reagent, such as bromoacetylbromide, to form the reactive bromoacetamide group which then readily alkylates free proteins to form the protein/macrocycle conjugate.
The noncovalent binding of these lanthanide element macrocycle compounds permits enhanced luminescence to be used with stains for proteins and other compounds. These stains can be used for electrophoresis including staining of gels that are predominantly composed of water. Other applications include fingerprint detection.
In a particularly preferred embodiment, a composition of the invention can include two different MMac each coupled to a polynucleotide as energy transfer acceptors, or two different MMac as energy transfer acceptors, each coupled to a different polynucleotide, and having luminescence enhanced according to the invention. When the different MMac differ in their central atoms, as with an europium macrocycle and a samarium macrocycle, and hence in emission peaks, measurement of the intensity of each peak provides a measure of the concentration of each MMac and of their relative ratios over a range from 500:1 to 1:500.
An important application of the above effect is the measurement of relative concentrations of normal cell DNA and cancer cell DNA by coupling each of these to a different MMac.
For further details of the coupling capabilities of functionalized macrocycles of Formula I reference can be made to Vallarino et al U.S. Pat. No. 5,696,240 at column 21 line 52 to column 22 line 42, here incorporated by reference.
When a functionalized macrocycle of Formula I is coupled directly or through a bridging/linking moiety to a reactive biomolecule, the resulting conjugate has the formula 
in which M, X, R, and n are as defined above; from one to two of A, B, C, and D are functionalized groups as defined above, and the remaining groups of A, B, C, and D are selected from the group consisting of hydrogen, straight-chain alkyl, branched-chain alkyl, aryl-substituted alkyl, aryl, and alkyl-substituted aryl; L is a bridging/linking moiety between the functionalized macrocycle and a biologically active compound, Z is a residue of a biologically active compound linked to L, a is zero or one, b is one, and each of f, g, h, and j is independently zero or one, provided that the sum of f, g, h, and j is either one or two.
When a functionalized macrocycle of Formula I is coupled to a bridging/linking moiety with the capability of further reacting with an analyte to form a conjugate, the resulting complex has Formula V in which L is a bridging/linking moiety capable of coupling the functionalized macrocycle and the analyte, a is one and b is zero, and M, X, R, n, A, B, C, D, f, g, h, and j are as defined above.
As a result of the ability of analytes including reactive biomolecules to bond to a functionalized macrocycle in a composition of this invention, as expressed by Z in Formula V, the enhanced luminescence of the composition can serve as an analytical tool for estimating such biomolecules as analytes. Thus the analyte can be any compound of interest, naturally occurring or synthetic, for which there exists a complementary binding partner.
These analytes are conveniently grouped by molecular weights. One group of such analytes consists of compounds that have molecular weights in the range of about 125-2,000 daltons and include a wide variety of substances, which are often referred to as haptens. These compounds include:
(a) Vitamins, vitamin precursors, and vitamin metabolites including retinol, vitamin K, cobalamin, biotin, folate;
(b) Hormones and related compounds including
(i) steroid hormones including estrogen, corticosterone, testosterone, ecdysone,
(i) aminoacid derived hormones including thyroxine, epinephrine,
(i) prostaglandins,
(i) peptide hormones including oxytocin, somatostatin,
(c) pharmaceuticals including aspirin, penicillin, hydrochlorothiazide,
(d) Nucleic acid constituents including
(i) natural and synthetic nucleic acid bases including cytosine, thymine, adenine, guanine, uracil, derivatives of said bases including 5-bromouracil,
(ii) natural and synthetic nucleosides and deoxynucleosides including 2-deoxyadenosine, 2-deoxycytidine, 2-deoxythymidine, 2-deoxyguanosine, 5-bromo-2-deoxyuridine, adenosine, cytidine, uridine, guanosine, 5-bromo uridine,
(iii) natural and synthetic nucleotides including the mono, di, and triphosphates of 2-deoxyadenosine, 2-deoxycytidine, 2-deoxythymidine, 2-deoxyguanosine, 5-bromo-2-deoxyuridine, adenosine, cytidine, uridine, guanosine, 5-bromouridine,
(e) drugs of abuse including cocaine, tetrahydrocannabinol,
(f) histological stains including fluorescein, DAPI
(g) pesticides including digitoxin,
(h) and miscellaneous haptens including diphenylhydantoin, quinidine, RDX.
Another group of analytes consists of compounds having a molecular weight of 2,000 daltons or more; including
(a) proteins and their combinations including
(i) albumins, globulins, hemoglobin, staphylococcal protein A, alpha-fetoprotein, retinol-binding protein, avidin, streptavidin, C-reactive protein, collagen, keratin,
(ii) immunoglobulins including IgG, IgM, IgA, IgE,
(iii) Hormones including lymphokines, follicle stimulating hormone, and thyroid stimulating hormone,
(iv) enzymes including trypsin, pepsin, reverse transcriptases
(v) cell surface antigens on T- and B-lymphocytes, i.e. CD-4, CD-8, CD-20 proteins, and the leukocyte cell surface antigens, such as described in the presently employed CD nomenclature;
(vi) blood group antigens including A, B and Rh,
(vii) major histocompatibility antigens both of class 1 and class 2,
(viii) hormone receptors including estrogen receptor, progesterone receptor, and glucocorticoid receptor,
(ix) cell cycle associated proteins including protein kinases, cyclins, PCNA, p53,
(x) antigens associated with cancer diagnosis and therapy including BRCA(s) carcinoembryonic antigen, HPV 16, HPV 18, MDR, c-neu; tumor surpressor proteins, p53 and retinalblastoma,
(xi) apoptosis related markers including annexin V, bak, bcl-2, fas caspases, nuclear matrix protein, cytochrome c, nucleosome,
(xii) toxins including cholera toxin, diphtheria toxin, and botulinum toxin, snake venom toxins, tetrodotoxin, saxitoxin,
(xiii) lectins including concanavalin, wheat germ agglutinin, soy bean agglutinin,
(b) polysialic acids including chitin;
(c) polynucleotides including
(i) RNAs including segments of the HIV genome, human hemoglobin A messenger RNA,
(ii) DNAs including chromosome specific sequences, centromeres, telomere specific sequences, single copy sequences from normal tissues, single copy sequences from tumors.
The biomolecule to be coupled to the macrocyclic complex for imaging or therapy is typically one selected to carry out a specific target function. In one embodiment, the biomolecule is a monoclonal antibody or antibody fragment which is specific against a selected cell-surface target site. Such antibodies are commercially available, or are made by well-known techniques.
In a preferred embodiment, the lanthanide element of the energy transfer acceptor macrocyclic compound is europium, samarium, or terbium. In a particularly preferred embodiment, a composition of the invention includes an energy transfer acceptor macrocyclic compound in which the central atom is europium and a second energy transfer acceptor macrocyclic compound in which the central atom is samarium. The characteristic emission peaks of europium and samarium in the spectrum are sufficiently separated so that the two macrocyclic compounds can be measured in the presence of one another. As a result, two different biomolecules can be measured in the presence of one another by using an enhanced luminescence composition of the invention whereby one is coupled to a functionalized europium macrocycle and another is coupled to a functionalized samarium macrocycle.
Also in accordance with this invention, the enhanced luminescence of the composition of the invention is produced by the interaction in an aqueous micelle organization of an energy transfer acceptor lanthanide element macrocycle compound as defined above with a luminescence-enhancing amount of at least one energy transfer donor compound of yttrium or a 3-valent lanthanide element having atomic number 59-71, preferably a compound of yttrium, lanthanum, or gadolinium. The energy transfer donor compound is ionic and soluble in water.
The energy transfer donor compound in the composition is present in a concentration greater than the concentration of the energy transfer acceptor macrocycle compound. The concentration of the energy transfer donor compound can range from 1xc3x9710xe2x88x925 to 1xc3x9710xe2x88x923 moles per liter.
In a preferred composition according to the invention, the energy transfer donor compound is an ionic compound of or complex of gadolinium (III). The gadolinium (III) halides and especially gadolinium (III) trichloride are particularly preferred.
The enhanced luminescence composition of the invention is preferably adjusted to a pH in the range from 5.5 to 8.5, suitably by use of a buffer system. The buffer system is preferably free of multivalent inorganic anions such as borate, carbonate, and phosphate that can cause precipitation of the energy transfer acceptor compound from the solution. Preferred buffer materials include hexamethylenetetramine and tricine, both of which are commercially available.
The enhanced luminescence composition of the invention exists in a micellar organization. The importance of micellar organization to the enhanced luminescence composition is demonstrated by the observation that a water-miscible polar solvent such as ethanol when added to the characteristically cloudy and luminous composition completely discharges the luminescence and simultaneously turns the cloudy micellar liquid clear. Once formed in an aqueous micellar organization, the composition of the invention can be transferred to an immiscible non-aqueous medium and/or dried, as by evaporation or lyophilization, with preservation of its luminescence. To provide the micellar organization, the composition includes a micelle-forming amount of a surfactant.
Surfactants (a coined term derived from xe2x80x9csurface active agentxe2x80x9d) are well known in the art and are organic compounds having a hydrophilic moiety and a hydrophobic moiety linked in the molecule. Surfactants are classified as amphoteric, anionic, cationic, or non-ionic according to the nature of the hydrophilic moiety; amphoterics include a hydrophilic anion, typically a carboxylate, sulfate, or sulfonate ion, and a hydrophilic cation, typically an ammonium ion; anionic and cationic surfactants include a hydrophilic ion of the respective type; and nonionic surfactants include an unionized hydrophilic group, typically a hydroxy(polyethylenoxy) group (CH2CH2O)pCH2CH2OH where p can range from 4 to about 30, preferably from 6 to 14, usually termed an xe2x80x9cethoxylatexe2x80x9d. Surfactants are further classified according to the hydrophobic moiety as alkyl, olefin, alkylbenzene, alkylphenol, polypropoxy etc.
Illustrative surfactants that can be used include cocoylamidopropylbetaine (amphoteric), household soap, lecithin, and dioctyl sodium sulfosuccinate (anionic), didodecyldimethylammonium chloride, cetylpyridinium bromide, and dodecylbenzylt-rimethylammonium chloride (cationic), glycerol monooleate, ethoxylated sorbitan monostearate, and ethoxylated nonylphenol (nonionic).
Cetyltrimethylammonium bromide, a cationic surfactant, is used in the preferred embodiment. The preferred concentrations for this surfactant range from 1.0xc3x9710xe2x88x924 to 1.0xc3x9710xe2x88x926 mol/L.
For a convenient compilation of many cationic surfactants reference can be had to McAtee U.S. Pat. No. 5,607,980 at column 7 line 55 to column 9 line 24, which disclosure is here incorporated by reference.
The concentration of surfactant is sufficient to form the micellar organization and thus typically exceeds the critical micelle concentration (CMC). The CMC has been measured and published for many surfactants. The suitable concentration of a surfactant whose CMC is not known is readily determined by incremental addition of the surfactant to a composition containing all the other intended ingredients until enhanced luminescence is observed.
In addition to the above disclosed energy transfer acceptor macrocycle compound, energy transfer donor compound, surfactant, and buffer ingredients, the composition of the invention can also contain one or more synergistic ligands to increase the luminescence of the composition beyond that attainable in absence of synergistic ligand. Such ligands do not displace the macrocycle of the acceptor or release the metal from the macrocycle and are presently believed to act by coordinating additional ligands to both acceptor (Eu or Sm or Tb and donor (Gd or Y, or La) in available spaces in the coordination sphere and thus prevent access of water that would cause vibrational quenching of the acceptor and/or donor.
Preferred synergistic ligands include trioctylphosphine oxide and 1,10-phenanthroline. The concentration of synergistic ligand when present can range from 10xe2x88x923 to 10xe2x88x926 moles/liter.
Moreover, the composition of the invention can contain one or more betadiketones. The concentration of betadiketone when present can range from 1xc3x9710xe2x88x922 to 1xc3x9710xe2x88x925 moles per liter. Preferred betadiketones have the formula RfCOCH2COQ in which Rf is a perfluoroalkyl group having 1 to 8 carbon atoms and Q is a carbocyclic or heterocyclic aromatic group or an alkyl group having 1 to 11 carbon atoms. A particularly preferred betadiketone is thenoyltrifluoroacetone.
The reaction medium in which a sample containing or suspected of containing an analyte is contacted with a functionalized macrocyclic complex according to this invention is preferably an aqueous solution in which the presence of foreign materials such as salts or organic solvents is limited to such concentrations as are tolerated by the analyte without denaturation, degradation, coagulation, hydrolysis, polymerization or other interfering changes. Binding conditions include such conditions of temperature, pressure, and pH as favor the reaction of the analyte with the functionalized macrocyclic complex, preferably a temperature in the range from 10xc2x0 C. to 45xc2x0 C., a pressure in the range from 800 to 1000 millibars, and a pH in the range from 5.5 to 8.5.
The functionalized metal complex according the method of the invention is characterized by kinetic stability even in very dilute aqueous solution. The complex is resistant to removal or exchange of the central metal atom, and has a counterion or balancing anion readily exchanged for other anions. The central metal atom is a lanthanide metal atom which can be yttrium (III), a 3-valent actinide atoms (atomic number 89-103), as well as a 3-valent central atom of a lanthanide elements of atomic number 57-71.
The functionalized metal complex according to the method of the invention is further characterized by the fluorescence spectrum with emission in the range from 500 to 950 nanometers upon excitation in the range from 200 to 400 nanometers.
In a preferred embodiment, the lanthanide element of the functionalized macrocyclic metal complex is europium, samarium, or terbium. In a particularly preferred embodiment, a functionalized macrocyclic metal complex of europium and a functionalized macrocyclic metal complex of samarium are used in combination in the method according to the method of the invention. The characteristic emission peaks of europium and samarium in the spectrum are sufficiently separated so that the two macrocyclic complexes can be measured in the presence of one another. As a result, two different analytes can be measured in the presence of one another by the method of the invention whereby one is coupled to a functionalized europium macrocycle complex and another is coupled to a functionalized samarium macrocycle complex.
The macrocycle of the functionalized metal complex according to the method of the invention has six coordinating atoms, of which at least 4 are nitrogen atoms, and the remainder are nitrogen, oxygen, or sulfur.
In a particularly preferred embodiment, the functionalized metal complex according to the method of the invention has the formula 
Wherein
M is a metal ion selected from the group consisting of a lanthanide having atomic number 57-71, an actinide having atomic number 89-103 and yttrium(II) having atomic number 39;
R is a substituent selected from the group consisting of hydrogen, straight-chain alkyl, or branched-chain alkyl; aryl-substituted alkyl, aryl, and alkyl-substituted aryl, with the proviso that such substituent does not limit the solubility of the resultant complex.
X is selected from the group consisting of nitrogen, sulfur and oxygen which forms a part of a ring structure selected from the group consisting of pyridine, thiophene or furan, respectively, at the positions marked X;
n is 2 or 3;
Y is a negatively charged ion, including acetate, carboxylate, sulfonate, halide, nitrate, perchlorate, thiocyanate, and picrate, with the proviso that such negative ion does not limit the solubility of the resultant complex or otherwise interfere with either the coupling procedure or the energy transfer leading to fluorescence;
m is the ionic charge of the metal ion in the macrocyclic complex, and;
yxe2x88x92 is the ionic charge of the counterion in the macrocyclic complex.
A, B, C, and D are substituents selected from the group consisting of hydrogen, straight-chain alkyl, or branched-chain alkyl; aryl-substituted alkyl, aryl, or alkyl-substituted aryl; reactive functionality, functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, or functionalized alkyl-substituted aryl;
at least one and not more than two of the substituents A, B, C, and D are selected from the group consisting of a reactive functionality, functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, or functionalized alkyl-substituted aryl, with the proviso that groups of said substituent provide coupling functionality between said substituent and a bridging/linking moiety to permit the derivatization thereof with a receptor molecule or an entity for which there is a corresponding receptor molecule;
and the remaining substituents A, B, C, and D are selected from the group consisting of reactive functionality, functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, or functionalized alkyl-substituted aryl, and groups that permit the coupling of the macrocycle to the biosubstrate, while also providing additional features such as increased solubility, greater stability, enhanced luminescence, or a combination thereof.
Straight chain and branched chain alkyl substituents at A, B, C, and/or D have from 1 to 25 carbon atoms. Reactive functionality signifies any substituent capable of reacting with a biologically active compound to form a covalent bond, such as alcoholic hydroxyl, phenolic hydroxyl, aldehyde, amino, carboxylic acid, carboxamide, isocyanate, isothiocyanate, mercapto and nitrile substituents. Functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, and functionalized alkyl-substituted aryl signify the respective groups substituted with a reactive functionality. Illustrative functionalized substituents include hydroxymethyl, 4-hydroxybenzyl, 4-aminobenzyl, and 4-isothiocyanatobenzyl.
Through these substituent groups, coupling can take place with an analyte. Such coupling can take place directly, as in a conjugate of a MMac with a protein or a polynucleotide linked to the MMac through a functionalized group at A, B, C, or D.
Coupling of a functionalized group at A, B, C, or D with an analyte can also take place indirectly, by reaction of the functionalized group and a bridging/linking moiety providing the capability for derivatization with a receptor molecule or an entity for which there is a corresponding receptor molecule together with controlled spacing of the analyte relative to the macrocycle of Formula I. Thus coupling is accomplished indirectly, either by the use of a bifunctional crosslinking reagent that provides covalent binding to the analyte, or by binding the macrocycle to another molecule that has a high affinity for the analyte. To illustrate, the bifunctional crosslinking reagent can be a protein or protein derivative capable of binding biotin, such as avidin or streptavidin, thus providing a link between biotin and the Mmac. In another illustrative reaction, an amine-functionalized macrocyclic complex of Formula I is acylated with a reagent, such as bromoacetylbromide, to form the reactive bromoacetamide group which then readily alkylates free proteins to form the protein/macrocycle conjugate.
When a functionalized macrocycle of Formula I is coupled directly or through a bridging/linking moiety to an analyte, the resulting conjugate has the formula 
in which M, X, R, and n are as defined above; from one to two of A, B, C, and D are functionalized groups as defined above, and the remaining groups of A, B, C, and D are selected from the group consisting of hydrogen, straight-chain alkyl, branched-chain alkyl, aryl-substituted alkyl, aryl, and alkyl-substituted aryl; L is a bridging/linking moiety between the functionalized macrocycle and an analyte, Z is a residue of a biologically active compound linked to L, a is zero or one, b is one, and each of f, g, h, and j is independently zero or one, provided that the sum of f, g, h, and j is either one or two.
When a functionalized macrocycle of Formula I is coupled to a bridging/linking moiety with the capability of further reacting with an analyte to form a conjugate, the resulting complex has Formula V in which L is a bridging/linking moiety capable of coupling the functionalized macrocycle and an analyte, a is one and b is zero, and M, X, R, n, A, B, C, D, f, g, h, and j are as defined above.
A variety of instruments is commercially available according to this invention for monitoring the presence and/or concentration of the conjugate of a functionalized macrocyclic metal complex with an analyte, the presence and/or concentration of the product of the interaction of a functionalized macrocyclic metal complex with a binding material; and the presence and/or concentration of the product of the interaction of the conjugate with the binding material.
Time-gated fluorescence instrumentation can be used according to this invention, while equally effective and less expensive fluorescence instrumentation equipped with a continuous as opposed to pulsed light source can now be used as a result of this invention. Such instrumentation can include a standard manual or automated fluorometer for reading samples. Also suitable is fluorescence instrumentation that measures multiple samples at a time, having a luminescence detection zone in which multiple samples can be automatically positioned. Such instrumentation can include a microtiter plate or strip positioning system.
Among preferred continuous light source fluorescence instruments of these types can be mentioned SPEX 1692T spectrofluorometer manufactured by Instruments SA Spex Fluorescence Division and LS-50B Luminescence Spectrophotometer manufactured by Perkin Elmer LLC, 761 Main Avenue, Norwalk, Conn. 06859-0010 USA.
In a particularly preferred type of fluorescence instrumentation, the instrument includes the capability to image the sample being analyzed, and especially to measure the analyte at various points in the image. This can be accomplished in particular as the instrument measures, records, processes, and/or displays the spatial distribution of one or more analytes. Instrumentation with these capabilities include Chromoscan manufactured by Applied Imaging Corporation 2380 Walsh Avenue, Santa Clara, Calif. 95051 and Axioplan 2 imaging manufactured by Carl Zeiss, Inc. One Zeiss Drive Thornwood, N.Y. 10594.
Particularly preferred applications of the method include comparative genomic hybridization and measurement of one or more samples for an analyte on a microarray.
In an important extension of the method of the invention, the enhanced fluorescence composition of the invention formed in an aqueous micellar organization can be dried and/or transferred into a non-aqueous medium and measured in the non-aqueous environment or in the dry state.
The following examples, of compositions characterized by kinetic stability and containing a metal compound resistant to removal or exchange of the central metal atom, and their use, are provided by way of illustration and not of limitation of the invention, whose scope is defined by the appended claims.